Novel immune response targeting molecules

ABSTRACT

The present invention provides methods of enhancing the immune response to an immunogen and to compositions for use in these methods. In particular the present invention provides a DNA molecule for use in raising an immune response to an antigen. The DNA molecule includes a first sequence encoding a targeting molecule, a second sequence encoding the antigen or an epitope thereof, and optionally a third sequence encoding a polypeptide which promotes dimerisation or multimerisation of the product encoded by the DNA molecule.

FIELD OF THE INVENTION

[0001] The present invention relates to methods of enhancing the immuneresponse to an immunogen and to compositions for use in these methods.In particular the present invention relates to the use of targetingmolecules in DNA and protein vaccination.

BACKGROUND OF THE INVENTION

[0002] The ability of direct injection of non-replicating plasmid DNAcoding for viral proteins to elicit protective immune responses inlaboratory and preclinical models has created increasing interest in DNAimmunisation. A useful review of DNA vaccination is provided in Donnellyet al, Journal of Immunological Methods 176 (1994) 145-152, thedisclosure of which is incorporated herein by reference.

[0003] Intramuscular injection of DNA as a means of vaccination caninduce both cellular and humoral responses (1). Studies using reporterproteins demonstrated that muscle cells are the principal target fortransfection after intramuscular DNA injection (2). The mechanismsunderlying the induction of immune responses after DNA immunisation areunclear. Since myocytes express MHC Class I at low levels and do notconstitutively express Class II or costimulatory molecules such as B-7(3), they appear unlikely candidates for the induction of Ab or CTLresponses. It is possible that low level transfection of antigenpresenting cells (APCs) occurs at the injection site and these APCs thentraffic to lymphoid organs and present the encoded antigen to B and Tcells (4) as has been shown after intradermal (5) and biolistic DNAimmunisation (6). Alternatively the myocyte may act merely as a sourceof antigen and priming occurs in the draining lymph node. In the lattercase, optimum immune induction would result if the antigen was releasedfrom the myocyte by secretion or subsequent to cell damage.

[0004] One strategy that has been shown to augment the response topolynucleotide, or DNA, vaccination is the use of sequences encodingcytokines or co-stimulatory molecules (Conry et al, (1996) Gene Therapy3: 67-74). These investigators showed an increased response when the DNAadministered encoded not only the antigen of interest but also for B7-1.

[0005] The present inventors investigated the effects of modifying theantigen such that it will be targeted to APC or sites of immuneinduction. This was shown to not only markedly enhance the immuneresponse but also cause immune deviation.

SUMMARY OF THE INVENTION

[0006] In a first aspect the present invention consists in a DNAmolecule for use in raising an immune response to an antigen, the DNAmolecule including a first sequence encoding a targeting molecule, asecond sequence encoding the antigen or an epitope thereof, andoptionally a third sequence encoding a polypeptide which promotesdimerisation or multimerisation of the encoded product.

[0007] As will be appreciated by those skilled in the art in a number ofinstances the antigen or epitope encoded by the second sequence will bea polypeptide which promotes dimerisation or multimerisation of theencoded product. As will be understood in such instances the thirdsequence may be omitted.

[0008] In a second aspect the present invention consists in apolypeptide, the polypeptide being encoded by the DNA molecule of thefirst aspect of the invention.

[0009] In a third aspect the present invention consists in a method ofraising an immune response in an individual, the method comprisingadministering to the individual the DNA molecule of the first aspect ofthe present invention or the polypeptide of the second aspect of thepresent invention.

[0010] There are a wide range of molecules which could be used astargeting molecules. These include ligands which target lymphoid cells(which will either be at or take the Ag to sites of immune induction),lymphoid sites (eg. spleen, lymph nodes, Peyers patches) or APCsdirectly. Examples of such ligands include, but are not limited to,CD40L, OX40, antibodies to receptors on APCs (eg. DEC 205, CD 23, CD11c,MHC class II), CD28, CTLA4 and L-selectin. It is presently preferredthat the targeting molecule is CTLA4 or L-selectin.

[0011] In a fourth aspect the present invention consists in a method ofdeviating the immune response to an antigen in an individual, the methodcomprising administering to the individual a DNA molecule including afirst sequence encoding CTLA4, a second sequence encoding the antigen oran epitope thereof, and optionally a third sequence encoding apolypeptide which promotes dimerisation or multimerisation of theencoded product.

[0012] There are many ways of producing dimerisation or multimerisationincluding tandem duplication and the use of any molecule that normallyforms multimers (e.g. Immunoglobulins, CD8, TNF, glutathiones-transferase, zinc finger dimers etc). There are many references in thescientific literature regarding this area. These include Classon B J etal (1992) “The hinge region of the CD8 alpha chain: structure,antigenicity, and utility in expression of immunoglobulin superfamilydomains” Int Immunol 4:215-25; Yang J, Moyana T, Xiang J (1995) “Agenetically engineered single-chain FV/TNF molecule possesses theanti-tumor immunoreactivity of FV as well as the cytotoxic activity oftumor necrosis factor.” Mol Immunol. 32:873-81; Tudyka T, Skerra A(1997) “Glutathione s-transferase can be used as a c-terminal,enzymatically active dimerization module for a recombinant proteaseinhibitor, and functionally secreted into the periplasm of Escherichiacoli. ” Protein Science. 6:2180-2187; Pomerantz J L, Wolfe S A, PaboColo. (1998) “Structure-based design of a dimeric zinc finger protein”Biochemistry 37:965-970; and Whiteheart S W, Rossnagel K, Buhrow S A,Brunner M, Jaenicke R, Rothman J E (1994) “N-ethylmaleimide-sensitivefusion protein: a trimeric ATPase whose hydrolysis of ATP is requiredfor membrane fusion.” J Cell Biol 126:945-54. The disclosure of thesereferences and the other references referred to in this application areincluded herein by cross-reference.

[0013] As will be appreciated by those skilled in the art in theconstructs of the present invention the first, second and third DNAsequences may be in any particular order. It is presently preferred thatthe order is first, third then second.

[0014] Throughout this specification, unless the context requiresotherwise, the word “comprise”, or variations such as “comprises” or“comprising”, will be understood to imply the inclusion of a statedelement or integer or group of elements or integers but not theexclusion of any other element or integer or group of elements orintegers.

DETAILED DESCRIPTION OF THE INVENTION

[0015] In order that the nature of the present invention may be moreclearly understood preferred forms thereof will now be described withreference to the following examples and Figures in which:

[0016]FIG. 1. Secretion of ΔIg, CTLA4Ig and L-SELIg proteins from NITtransfectants. NIT cells were transfected with the pRep10::CD5L-hIg,pRep7::mCTLA4-hIg and pRep10::hL-SEL-hIg expression plasmids. Secretedprotein was purified on immobilised protein A and samples run by SDSPAGE under reducing and non-reducing conditions.

[0017]FIG. 2. hIg specific IgG responses in DNA immunized mice. Serawere obtained from BALB/c mice immunized with pRep10::CD5L-hIg,pRep7::mCTLA4-hIg and pRep10::hL-SEL-hIg at the indicated times postimmunisation and stored at −20° C. until assayed for hIg specific IgG inan ELISA. Titres were defined as the highest dilution to give a 0.2 ODat 450 nm. Results are expressed as the mean of the log titre ±SEM from5 mice in each group. Normal mouse sera and hyperimmune mouse seraserved as the negative and positive controls respectively.

[0018]FIG. 3. hIg specific IgG subclass responses in DNA immunized mice.A. Sera were obtained from BALB/c mice immunized with pRep10::CD5L-hIg,pRep7::mCTLA4-hIg and pRep10::hL-SEL-hIg at 8 weeks post immunisationand stored at −20° C. until assayed for hIg specific IgG1, IgG2a orIgG2b in an ELISA. Titres were defined as the highest dilution to reachan OD of 0.2 at 450 nm. Results are expressed as the mean of the logtitre ±SEM from 5 mice in each group. B. The log IgG1 titre for eachmouse was divided by the corresponding log IgG2a titre to obtain a logIgG1: log IgG2a ratio. Results are expressed as the mean ±SEM from 5mice in each group.

[0019]FIG. 4. hIg specific IgG subclass responses in soluble proteinimmunized mice. Sera were obtained from BALB/c mice immunized with 5 μgof hIg or 5 μg of CTLA4Ig protein in 100 μl of PBS, 2 weeks postimmunisation and assayed for hIg specific IgG in an ELISA. Titres weredefined as the highest dilution to reach an OD of 0.2 at 450 nm. Resultsare expressed as the mean of the log titre ±SEM from 5 mice in eachgroup.

[0020]FIG. 5. hIg specific IgG subclass responses in CTLA4Ig DNAimmunized mice. Sera were obtained from BALB/c mice immunized with theindicated dose of pRep7::mCTLA4-hIg 2 weeks post immunisation andassayed for hIg specific IgG1, IgG2a or IgG2b in an ELISA. Titres weredefined as the highest dilution to reach an OD of 0.2 at 450 nm. Resultsare expressed as the mean of the log titre ±SEM from 5 mice in eachgroup.

[0021]FIG. 6. OVA specific IgG and IgG subclass responses afterco-injection of DNA. Sera were obtained from BALB/c mice immunized withpRep10::hL-SEL-hIg and pCI-OVA or pRep7::mCTLA4-hIg and pCI-OVA at 4weeks post immunisation and assayed for OVA specific IgG (A) or IgG1,IgG2a or IgG2b (B) in an ELISA. Titres were defined as the highestdilution to reach an OD of 0.2 at 450 nm. Results are expressed as themean of the log titre ±SEM from 5 mice in each group.

[0022]FIG. 7. Shows stimulation index with hIg, Lsel-hIg and CTLA4-hIg(HuIg 1mg/ml;

1HuIg 1 mg/ml; HuIg 1 mg/ml)

[0023]FIG. 8. Shows anti-ovalbumin IgG titres with various constructs (

2 weeks; 4 weeks)

[0024]FIG. 9. Shows anti-OVA IgG titres with pCI::mCTLA4-g3h-OVA andpCI::mCTLA4-hIg-OVA

[0025]FIG. 10. Groups of 5 Balb/c mice were vaccinated intramuscularlyon days 0 and 28 with 0.1 mg of pCI::mCTLA4-hIg-45W (black circles) orpCI::CD5L-hIg-45W (grey circles). Mice were bled on days 0, 7, 14, 28,35 and 42. Sera was assayed for anti-45w antibodies by ELISA usingrecombinant 45W(His)6. The Student's t-test was used to compared the twogroups and the probability values (P) for the two vaccines at each timepoint are shown at the top of the figure.

[0026]FIG. 11. Groups of 5 Balb/c mice were vaccinated eitherintraperitoneally with 20 μg of recombinant 45w(His)6 protein (greycircles ) in Freund's complete adjuvant or intramuscularly with 0.1 mgpCI::mCTLA4-hIg-45W (black circles) in 0.1 ml of saline. Mice were bledon days 0, 2 ,5 ,8 , 14 and 28 post-vaccination and anti-45w antibodiesmeasured by ELISA using 45W(His)6 protein. The responses were comparedby Student's t-test and were different at day 8 (p<0.05).

[0027]FIG. 12. Shows % survival of mice following challenge withPlasmodium chabaudi adami DS (pCI::CD5LhIg-AMA; pCI::CTLA4-hIg-AMA;

pCI::CTLA4-hIg).

[0028]FIG. 13 shows antibody titres.

[0029]FIG. 14 shows antibody titres at day 14 with varying immunisations

[0030]FIG. 15 shows antibody titres at day 54 with varying immunisations

[0031]FIG. 16 shows lung virus titres

[0032]FIG. 17. Kinetics of induction of the anti-hIg antibody titrespost-immunisation. Filled symbols represent plasmids containing hIg:filled squares (pCI::bCTLA4-hIg-ΔPLD), filled circles(pCI::CD5L-hIg-ΔPLD), filled triangles (pCI::bCTLA4-hIg). Open symbolsrepresent control animal groups not injected with hIg in any form: opencircles (pCI::ΔPLD), open squares (Unvaccinated controls) and opentriangles (Glan-Vac).

[0033]FIG. 18. Western Blot of PLD expressed by eukaryotic andprokaryotic cells. Lane 1-3. Supernatant from Cos-m6 cells transfectedwith pCI::PLD (lane 1), pCI::ΔPLD (lane 2) and pCI alone (lane 3). Lane4. Cell filtrate containing PLD expressed from Corynebacteriumpseudotuberculosis

[0034]FIG. 19. Protection from challenge with Corynebacteriumpseudotuberculosis. Percentage of the animals protected from challengeby 10⁶ CFU of Corynebacterium pseudotuberculosis injected just above thecoronet. Protection was defined as the animal not having abscesses inany of the following lymph nodes: popliteal, inguinal and prefemoralboth left and right.

[0035]FIG. 20 Shows number of mice with lesions at various time pointsafter challenge with L. major (□ PBS; ▴ pCI::CD5L-hIg-PSA2; ▪pCI::mCTLA4-hIg-PSA2; Δ pCI::PSA2)

EXAMPLE 1 Materials and Methods

[0036] Mice

[0037] Female mice (BALB/c, CBA and C57B1/6) aged 6 to 8 weeks were usedin all experiments. Mice were maintained in SPF conditions.

[0038] Plasmids and Immunisations

[0039] Expression plasmids were constructed to produce secreted forms ofthe Fc fragment of human IgG1 (ΔIg) by using the Cd5 leader sequence(CD5L) either alone or fused with murine CTLA4 (mCTLA4Ig) or humanL-selectin (hL-SELIg) under the control of the RSV promoter in the Rep7or Rep10 vectors (these vectors differ only in the direction of themultiple cloning site, Invitrogen, San Diego, Calif., U.S.A). Thesequence of pREP7::CTLA4-hIg is shown in Sequence ID No. 1 -PromoterRSV: 13-640, CTLA4-hIg: 703-2462. The constructs were obtained fromplasmids given by Drs. P. Lane (Basel Institute, Switzerland), B. Seed(Massachusetts General Hospital, Boston, USA) and D. L. Simmons(Institute of Molecular Medicine, Oxford, UK). The following constructswere generated:

[0040] pRep10::CD5T-hIG

[0041] pRep7::mCTLA4-hIg

[0042] pRep10::hLSEL-hIg

[0043] Plasmids for injection were prepared from E. coli by PEGprecipitation as described (7) except that volumes of Solution I, II andIII were adjusted such that pellets were resuspended in 50 mL ofSolution I for each Litre of broth media used. Endotoxin was removedfrom plasmid preparations by four Triton X-114 phase separations (8) andDNA was stored at −20° C. in normal saline until injected. The resultantplasmid preparations contained less than 10 IU endotoxin per mg ofplasmid DNA as determined by the limulus amoebocyte lysate assay(QCL-1000 BioWhittaker, Walkersville, Md., U.S.A). Mice received 100 μgof plasmid DNA in both quadriceps or intradermally at the base of thetail on day 0 and 14 of each experiment.

[0044] Antibody Assays

[0045] Microtitre plates (Dynatech, Chantilly, VI., USA) were coatedwith human Ig (hIg) protein (Intragam, CSL, Parkville, Australia; 10μg/ml in PBS) by overnight incubation at 4° C. and washed four timeswith PBS to remove unbound antigen. Plates were incubated with seriallydiluted sera in blocking buffer (5 % milk powder in PBS) overnight at 4°C. After washing 5 times with PBS to remove unbound Ab, plates wereincubated with peroxidase conjugated anti-mouse IgG, IgG1, IgG2a orIgG2b antibodies (Southern Biotechnology, Birmingham, Ala., USA) dilutedin blocking buffer. After washing five times with PBS, the amount ofbound Ab was determined by addition of substrate solution (0. 1 mg/ml3,3,5,5-tetra methylbenzidine (T2885, Sigma St. Louis, Mo., USA) 0. 03%H₂O₂ in 0.1M Na acetate pH6.0). The reaction was stopped with 1M H₂SO₄and the OD read at 450 nm. Titres were defined as the highest dilutionto reach an OD of 0.2.

[0046] To calibrate the IgG subclass ELISA, plates were coated withIgG1, IgG2a or IgG2b from mouse myelomas (10 μg/ml in 0.5 times PBS)overnight at 4° C., washed 3 times with PBS and then incubated withserially diluted anti-mouse IgG subclass HRP conjugated Ab. The dilutionof each anti-mouse subclass Ab which gave identical absorbances in theELISA were used subsequently.

[0047] Results and Discussion

[0048] Expression plasmids were constructed to produce secreted forms ofthe human IgG1 heavy chain alone (pRep10::CD5L-hIg) or fused with CTLA4(pRep7::mCTLA4-hIg) or L-selectin (pRep10::hLSEL-hIg). Cells transfectedwith these plasmids secreted the three molecules as disulphide linkeddimers of expected size (FIG. 1). Like others (1) we were unable todetect in vivo protein expression by western blotting of musclehomogenates and or of protein A purified material from sera of B-celldeficient immunized mice (data not shown). However, the ability todetect immune responses in immunized mice is indicative of in vivoexpression. No immune responses to human Ig were detected in unimmunisedor mice receiving vector only (data not shown). However, mice immunizedwith pRep10::CD5L-hIg, pRep10::hL-SEL-hIgΔIg or pRep7::mCTLA4-hIg hadmarkedly different responses (FIG. 2). Responses in pRep7::mCTLA4-hIgimmunized mice were more rapid and of greater magnitude at all threetime points: 2, 4 and 8 weeks (FIG. 2). At 4 weeks both thepRep7::mCTLA4-hIg and pRep10::hLSEL-hIg immunized mice had 1000 and 100fold higher IgG responses than pRep10::CD5L-hIg controls respectively.The differences observed were not attributable to any adjuvant effectsof endotoxin, because Triton X-114 was used to remove endotoxin (8) sothat the levels were <10IU/mg plasmid DNA. Similar results have beenachieved in 3 experiments using BALB/c and CBA mice (data not shown).

[0049] The response in all mice to pRep10::CD5L-hIg was dominated byIgG2a (FIG. 3), which mimics a viral infection, and has been reportedfor other antigens after DNA immunisation (9, 10). The IgG subclassresponse in pRep10::hLSEL-hIg immunized mice was similar (althoughgreater) to pRep10::CD5L-hIg controls whereas the pRep7::mCTLA4-hIgresponse was deviated to an IgG1 dominance (FIG. 3B). The possibilitythat the differences in Ab responses was due to dose was unlikely sinceall constructs were made with identical plasmid backbone and miceimmunized with soluble CTLA4Ig protein (FIG. 4) had higher Ab responsesthan those receiving an equivalent dose of hIg. Also, to determine ifthe IgG1 dominance of the response to pRep7::mCTLA4-hIg was due to dosewe immunized mice with different amounts of pRep7::mCTLA4-hIg so thatmice with total IgG antibody levels could be compared to that ofpRep10::hLSEL-hIg (FIGS. 2 and 3 ). The IgG1 predominance was found atall doses of pRep::mCTLA4-hIg (FIG. 5).

[0050] Work with CTLA4 has demonstrated it can bind to B-7 and blockco-stimulation which reduces the response to other immunogens (11). Anon-specific immunomodulatory effect of CTLA4 was unlikely for severalreasons. Firstly, CTLA4Ig protein at least in high doses (and hence B-7is blocked) has been ascribed immunosuppressive properties notimmunostimulating ones (11) as we found for DNA and proteinimmunisations. Furthermore, mice co-injected with CTLA4Ig and DNAencoding ovalbumin (pCI-OVA) had similar ovalbumin specific IgG and IgGsubclass titres to control mice (FIG. 6) indicating that there was notany immunosuppressive effect of CTLA4.

EXAMPLE 2 Use of Targeting Ligand to Augment T Cell ProliferativeResponses and Requirement for Dimerisation

[0051] Introduction

[0052] In Example 1 there is a demonstration that Ab levels to a modelDNA vaccine could be enhanced when antigen was fused with the targetingligands CTLA4 or L-selectin.

[0053] The hIg component would ensure dimerisation which we thoughtwould be favourable because in general, binding of ligands to receptorsis stronger when dimers are used. However, it was unclear in this systemif dimerisation of the antigen targeting ligand fusion proteins wasnecessary for increased immune responses. To determine if the enhancedAb response generated by antigen targeting vectors encoding proteins wasdependent upon dimer formation, Ab responses were compared toimmunisation with plasmid encoding monomeric antigen targeting ligandfusion proteins. The hIg component of the vectors was replaced withcoding sequence for another model antigen that would not form dimers(ovalbumin; OVA).

[0054] Materials and Methods

[0055] Female mice aged 6 to 8 weeks were used in all experiments andmaintained in SPF conditions.

[0056] After PCR amplification to include an Mlu I restriction enzymerecognition sequence, the OVA cDNA (bp 470-1170) was inserted behind thehuman immunoglobulin Fc (hIg) gene via a 4 amino acid glycine linker atthe Nsi I site. These vectors would form dimers due to the interchaindisulfide bonds of hIg and are represented by an hIg-OVA suffix. Atargeting vector that would not form dimers was obtained by directfusion of the cDNA from OVA to the cDNA of CTLA4 (pCI::mCTLA4-OVA) or tothe leader sequence of CD5 as a control (pCI::CD5L-OVA). After PCRamplification to include Hind III and Nsi I restriction sites the entirehIg component of pCI::mCTLA4-hIg-OVA was replaced with the human IgG3hinge region (a gift from Dr Y Akahori, Japan) to formpCI::mCTLA4-g3h-OVA. Plasmids for injection were prepared from E. coliwith endofree QIAGEN maxi kits according to the manufacturer'sinstructions and stored at −20° C. in normal saline until injected. Micereceived 50 μg of plasmid DNA in 100 μl normal saline i.m. in bothquadriceps at day 0 of each experiment.

[0057] The proliferation of 2×10⁵ splenocytes was determined by astandard 5 day ³H-thymidine uptake protocol at 6 weeks post initialimmunisation. The mean stimulation index was calculated as the cpm withantigen/cpm splenocytes alone.

[0058] Microtitre plates (NUNC, Maxisorb) were coated with OVA protein(A-5503, Sigma, St. Louis, Mo.; 10 μg/ml in PBS) by overnight incubationat 4° C. and washed four times with PBS to remove unbound antigen.Plates were incubated with serially diluted sera in blocking buffer (1 %casein in PBS) overnight at 4° C. After washing 5 times with PBS toremove unbound Ab, plates were incubated with peroxidase conjugatedanti-mouse IgG (Southern Biotechnology, Birmingham, Ala.) diluted inblocking buffer. After washing five times with PBS, the amount of boundAb was determined by addition of tetramethylbenzidine substratesolution. The reaction was stopped with 1M H₂SO₄ and the OD read at 450nm. Titres were defined as the highest dilution to reach an OD of 0.2.

[0059] Results and Discussion

[0060] The proliferation splenocytes was determined by a standard 5 day3H-thymidine uptake protocol at 6 weeks post initial immunisation (FIG.7). The stimulation index was calculated as the cpm with antigen/cpmsplenocytes alone. The mean ±SD from 3 mice in each group is shown afterincubation with three different antigen concentrations. Mice immunizedwith the DNA constructs pCI::mCTLA4-hIg and pCI::Lsel-hIg had 8 and 3fold higher T cell proliferative responses than controls (pCI::CD5L-hIg)respectively . This data suggested that the targeting of antigen washaving an enhancing effect on T cell activation.

[0061] Groups of 8 mice were immunized with DNAs expressing themonomeric targeting vector pCI::mCTLA4-OVA, the monomeric controlpCI::CD5L-OVA, or the dimeric vectors pCI::CD5L-hIg-OVA (control),pCI::Lsel-hIg-OVA or pCI::CTLA4-hIg-OVA on day 0 and bled 2 and 4 weekspost immunisation. The OVA specific IgG levels were determined by ELISA.The results obtained at 2 and 4 weeks post immunisation are illustratedin FIG. 8 (2 weeks; hatched columns, 4 weeks; solid columns). There wasno difference in Ab levels at 2 or 4 weeks between the mice immunizedwith pCI::CD5L-OVA or pCI::mCTLA4-OVA monomeric DNA vectors. The highestAb responses were obtained with the pCI::mCTLA4-hIg-OVA vector, whichforms dimers, compared to the monomeric (pCI::CD5L-OVA) or dimeric(pCI::CD5L-hIg-OVA) controls.

[0062] Surprisingly, the pCI::Lsel-hIg-OVA immunized mice had thepoorest responses at both time points. This data is in contrast to theenhanced responses to hIg when fused with L-selectin alone. Theobservation that the responses obtained with pCI::Lsel-hIg-OVA weresimilar in magnitude to those obtained with the monomeric antigenfusions suggests that the fusion of OVA (or other antigens) to Lsel-hIgmay interfere with the efficiency of binding of L-selectin to its ligand(e.g. by interfering with dimerisation, by allosteric effects or byconformational changes to L-selectin). Alternative ways of fusion shouldbe investigated.

[0063] Overall, these results suggest that for effective antigentargeting the inclusion of a molecule such as hIg that facilitatesdimerisation is essential unless the antigen itself facilitatesdimerisation or multimerisation. This data was obtained using the hinge,CH2 and CH3 domains of human IgG1. The dimerisation of hIg isfacilitated by disulfide bonds between cysteine residues in the hingedomains. To determine if another molecule that would also facilitatedimerisation could replace the hIg component, the hinge region of humanIgG3 was used to link mCTLA4 with OVA. Groups of 8 mice were immunizedwith DNAs expressing the targeting vectors pCI::mCTLA4-hIg-OVA orpCI::mCTLA4-g3h-OVA. At 2 weeks post immunisation sera was collected andshown to contain similar levels of anti-OVA antibodies (FIG. 9).Therefore, this suggests that it may be possible to reduce the hIgcomponent of the antigen targeting vectors to a hinge region alone orreplace the hIgG1 component with another immunoglobulin hinge region,another molecule or part thereof such as the hIgG3 hinge to facilitatedimerisation. This would be of particular applicability when thetargeting ligand is L-selectin or another molecule that does notdimerise or is structurally compromised by fusion of antigen via hIg.

[0064] Example 1 demonstrated an increased immune responses to hIg afterDNA and protein immunisation was obtained with targeting ligand-hIgfusions. The following Examples were conducted to determine if antigensother that hIg could be used for increased immune responses. These datawere obtained by the addition of antigens to the C-terminus of hIg whichcould facilitate dimer formation as was found with hIg alone. Agly-gly-gly-gly-thr spacer was introduced between hIg and the antigens.Whilst these constructs have been used it will be appreciated thatresponses may be improved by routine optimisation. This optimisation mayinvolve modification of the constructs as envisaged within the presentinvention eg different targeting molecules, different sequences whichfacilitate multimerisation, different linkers etc.

EXAMPLE 3 Use of CTLA4 to Accelerate Immune Responses Against the HostProtective Antigen of Taenia ovis Known as 45W

[0065] Introduction

[0066] The 45W antigen is a putative membrane glycoprotein present in,or underlying the tegument of, the Taenia ovis oncosphere. T. ovis is apathogen of sheep which causes commercial losses of mutton and wool inNew Zealand and other important sheep growing countries. Earlyimmunisation studies using 45W protein partially purified from T. ovisrevealed that it was a promising vaccine antigen. Subsequent fieldtrials using recombinant forms of 45W, expressed in Escherichia coli, asa vaccine reported very high levels (about 95%) of protection¹³. The 45Wantigen was used as a DNA vaccine in sheep and low levels of antibody,measured using a recombinant form of 45W, was observed¹⁴.

[0067] Materials and Methods

[0068] Plasmids containing the CMV promoter and the genes encodingCTLA4, the Fc portion of human immunoglobulin (hIg) and the CD5 signalpeptide were described above. The gene encoding 45W was obtained fromDr. Marshall Lightowlers (Dept. Veterinary Science, University ofMelbourne).

[0069] Inbred Balb/c mice of 6-8 weeks of age were obtained from theDept. Microbiology and Immunology Animal House, University of Melbourne.

[0070] Standard DNA manipulation and CsCl purification techniques wereused. The gene encoding 45W was ligated into two DNA vaccines. ConstructpCI::mCTLA4-hIg-45W expressed a fusion protein which comprised the CTLA4signal peptide, mouse CTLA4 ectodomain, hIg and the 45W antigen.Construct pCI::CD5L-hIg-45W expressed a fusion protein which containedthe signal peptide from CD5, hIg and the 45W antigen.

[0071] DNA (100 μg) was injected into the quadriceps of mice on days 0and day 28. Sera was obtained at appropriate intervals post vaccinationand analysed for total antibodies specific for recombinant 45W antigenin a titration ELISA¹⁵ using horseradish peroxidase conjugatedanti-mouse immunoglobulins.

[0072] Purified recombinant 45W(His)₆ was obtained from E. coliaccording to Rothel et al.¹⁴ using a polyhistidine ‘tag’ and nickelaffinity chromatography.

[0073] Results

[0074] Mice were immunised with the DNA vaccines pCI::mCTLA4-hIg-45W, orpCI::CD5L-hIg-45W (FIG. 10). Mice received 100 ug of DNA on days 0 and28 and were bled at weekly intervals. Mice receiving the DNA vaccinewhich expressed CTLA4 fused to the hIg/45W vaccine developed a morerapid antibody response than the mice which received a similar plasmidvaccine construct ie. pCI::CD5L-hIg-45W which did not contain the CTLA4gene. The mice receiving the vaccine with CTLA4 produced serumantibodies of high titre (ie.≦10,000) on days 7, 14 and 28. Incomparison, mice which received the construct lacking CTLA4 did notproduce high titre antibodies (ie. titre≦10,000) until after the secondimmunisation on day 28. All mice(ie. {fraction (5/5)}) which receivedthe DNA vaccine containing CTLA4 produced 45W-specific antibodies by 7days post immunisation whereas only ⅕ animals which received theequivalent DNA vaccine lacking CTLA4 produced antibodies at day 7 postimmunisation. The data was analysed using Student's t-test.

[0075] A second trial was undertaken where mice received either 20 μg ofpurified recombinant 45W(His)₆ protein in Complete Freund's Adjuvant, orthe CTLA4 DNA vaccine (ie. pCI::mCTLA4-hIg-45)(FIG. 11). The serumantibody response was examined on days 0, 2, 5, 8, 14 and 28. The serumantibody response specific for 45W was higher at day 8 in DNA vaccinatedmice than in mice which received the 45W protein vaccine.

[0076] Discussion

[0077] The murine serum antibody response to 45W DNA vaccination wasaccelerated by fusion of CTLA4 to the hIg-45W fusion protein. Theantibody response to 45W correlates with protection in sheep against T.ovis disease. Addition of CTLA4 led to a more rapid high titre response,with a shorter unprotected period following immunisation. The effect ofCTLA4 on the magnitude of the anti-45W response was not as dramatic asthe effect on human Ig described above. This may have been due to theconformational restraints from the fusion of the various molecules orsome inherent property of the 45W antigen. Furthermore, the immunisationprotocol employed in this Example differed from Example 1 in thatboosting occurred at 4 rather than two weeks. Due to the more rapidkinetics of the response via CTLA4 targeting boosting may not have beenoptimum and thus the magnitude was not effected.

EXAMPLE 4 Use of CTLA4 with AMA1 to Protect Against Plasmodium chabaudiadami in Mice

[0078] Introduction

[0079] AMA1¹⁶ is a candidate vaccine antigen against malaria. We haveevidence that domain3 of AMA1 folds independently and as such may be agood candidate in producing a fusion protein with hIg. However, althoughAMA1 has been shown to confer protection in mouse malaria¹⁷, we areunaware of any work that has shown domain3 to be protective.

[0080] Materials and Methods

[0081] Plasmids containing the CMV promoter and the genes encodingCTLA4, the Fc portion of human immunoglobulin (hIg) and the CD5 signalpeptide were described above. Domain 3 of AMA-1 from the Plasmodiumchabaudi adami DS strain¹⁶ was fused to CTLA4Ig and CD5LIg(pCI::mCTLA4-hIg-AMA and pCI::CD5L-hIg-AMA). The plasmid pCI::mCTLA4-hIgwas used as a negative control.

[0082] Inbred female Balb/c mice of 6-8 weeks of age were used.

[0083] DNA (100ug) was injected into the quadriceps of mice on day 0only. Mice were challenged with Plasmodium chabaudi adami DS and thenumber of deaths recorded.

[0084] Antibody titres were measured by ELISA using refolded E. coliexpressed entire ectodomain of AMA1¹⁷. The titres were expressed as thelog of the reciprocal of the last serum dilution to give an OD>0.1.

[0085] Results

[0086] In the first trial, there were 8 mice per group. A singleimmunisation with the DNA vaccine pCI::mCTLA4-hIg-AMA afforded partialprotection against an intraperitoneal challenge of 100,000 parasites(FIG. 12). Challenge was performed 14 days after immunisation. This wassignificant (log rank test; p<0.05) from the control pCI::mCTLA4-hIggroup. There was a clear indication that the CTLA4 conferred betterprotection than the pCI::CD5L-hIg-AMA group, although this did not reachstatistical significance. The antibody titres (FIG. 13) show that theCTLA4 targeting ligand enhances the antibody response to AMA1 (p<0.005).

[0087] A second trial was undertaken with larger group (16/group) andwith an intravenous challenge of 10,000 parasites. No protection wasseen in this second trial.

[0088] Discussion In the first trial, CTLA4 conferred some protectionagainst malaria by domain3 of antigen AMA1. This was not found in thesecond trial. We do not know why there was a difference between the twotrials. Because there is not a full set of antibody data for comparison,we do not know whether the level of antibody achieved was sufficient inthe mice that died in trial two, or whether the effect was due thedifferent route of challenge. We also do not know how effectiveCTLA4IgAMA may be when booster doses are given.

EXAMPLE 5 Use of CTLA4 in Influenza Infection in Mice

[0089] Introduction

[0090] As an additional model for testing protective efficacy we haveused influenza infection of the murine respiratory tract. The influenzahaemagglutinin (HA) gene was cloned behind targeting molecules and theresulting DNA vaccines examined for their ability to generate higheranti-viral antibody titres and afford greater protection against liveviral challenge compared to control vaccines not expressing thetargeting molecule.

[0091] Materials and Methods

[0092] Virus

[0093] The type A influenza virus used in this study was PR8=A/PuertoRico/8/34 (H1N1). Virus was grown in the allantoic cavity of 10-dayembryonated hens' eggs for 2 days at 35° C. The allantoic fluid wascollected and clarified by centrifugation (2000 g, 15 mins, 4° C.).Aliquots of allantoic fluid containing infectious virus were stored at−70° C. and used for immunisation and challenge of mice. Purified PR8virus used in ELISA assays was obtained as zonally purified stocks fromCSL Ltd, Parkville, Victoria, Australia. The haemagglutination assay¹⁸was used to quantitate virus and titres are expressed inhaemagglutinating units (HAU) per ml.

[0094] Immunisation Groups of 10 BALB/c mice were immunisedintramuscularly (i.m.) under anaesthesia on day 0 with 0.1 ml of DNAvaccine containing 50 μg of plasmid. The constructs used forimmunisation were pCI::CD5L-hIg-HA, pCI::mCTLA4-hIg-HA, which were basedon PR8 HA lacking the signal and transmembrane sequences, andpCI::CD5L-hIg-SIINFEKL (expressing an 8 amino acid sequence from chickenovalbumin) as a negative control. As a positive control, a group of 10BALB/c mice were infected intranasally (i.n.) with 50 plaque formingunits (pfu) of infectious PR8 virus and another group immunisedsubcutaneously with 1 μg of β-propiolactone (BPL)-inactivated and sodiumtaurodeoxycholate-disrupted PR8 virus (split virus). Serum samples werecollected from all mice on days 7, 14 and 54 and mice were thenchallenged on day 65.

[0095] Intranasal Challenge of Mice and Preparation of Mouse LungExtracts

[0096] Penthrane anaesthetised mice were challenged with 50 pfu ofinfectious PR8 influenza virus i.n.. Each mouse received 50 μl of virusin the form of allantoic fluid diluted in phosphate buffered saline(PBS). Five days after challenge, mice were killed by cervicaldislocation and lungs were removed and transferred aseptically tobottles containing 1.5 ml Hanks Balanced Salt Solution (HBSS),supplemented with 100 U of penicillin per ml, 100 μg of streptomycin perml and 30 μg of gentamicin per ml. Lung homogenates were prepared usinga tissue homogeniser. Each lung suspension was then centrifuged at 300×gfor 5 minutes and the supernatants were removed, aliquoted and stored at−70° C. prior to assay for infectious virus.

[0097] Enzyme-Linked Immunosorbent Assay

[0098] The enzyme-linked immunosorbent assay (ELISA) was performed aspreviously described by Jackson et al.¹⁹ using 96-well polyvinylmicrotitre trays (Dynatech, Australia) coated with a solution containing50 HAU of purified PR8 virus per well. Antibody titres are expressed asthe reciprocal of the antibody dilution giving an absorbance of 0.2units.

[0099] Plaque Assay for Infectious Virus

[0100] Virus titres were determined by plaque assay on monolayers ofMadin-Darby canine kidney (MDCK) cells in 35 mm petri dishes (Nunc,Roskilde, Denmark). The culture medium was RPMI-1640 supplemented with 2mM glutamine, 2 mM sodium pyruvate, 100 U of penicillin per ml, 100 μgof streptomycin per ml, 30 μg of gentamicin per ml and 10% (vol/vol)foetal calf serum (heat inactivated at 56° C. for 30 min). Monolayerswere washed with serum-free RPMI-1640 containing antibiotics andinoculated in duplicate with 100 μl of dilutions of lung homogenates inthe same medium. After allowing 45 minutes in a humidified incubator(37° C., 5% CO₂) for virus adsorption, 3 ml of agarose overlay medium at45° C. was added. Incubation was continued for a further 3 days andplaques counted. The agarose overlay medium was Leibovitz L-15 medium pH6.8 (Gibco Laboratories, U.S.A.) supplemented with 100 U penicillin perml, 100 μg of streptomycin per ml, 0.01 M HEPES buffer pH 6.8(Calbiochem, Australia), 0.1 % trypsin-TPCK (Worthington, BiologicalSystems Inc., U.S.A.) and 0.9% agarose (ICN Biomedicals Sydney,Australia).

[0101] Statistical Analysis

[0102] The data were analysed using the nonparametric Mann-Whitney Utest which compares two sets of unpaired samples. The null hypothesis isthat the two population medians are equal and the resultant P value forparticular comparisons is given.

[0103] Results and Discussion

[0104] Serum Antibody Responses of Mice

[0105] Sera collected from mice immunised with the DNA constructs, splitPR8 virus in PBS, or infectious PR8 virus were assayed for anti-viralantibody by ELISA. On day 7 after priming, only mice immunised withinfectious PR8 virus exhibited anti-viral antibody titres significantlyhigher than the background titres detected in mice given the control DNAconstruct pCI:: CD5L-hIg-SIINFEKL (p=0.0002). However by day 14, inaddition to the high antiviral antibody titres detected in the virusinfected mice, antibody titres of mice immunised with thepCI::mCTLA4-hIg-HA construct were significantly higher than those ofmice given the control DNA constructs pCI::CD5L-hIg-HA (p=0.0003) orpCI::CD5L-hIg-SIINFEKL (p=0.0004) (FIG. 14). Furthermore, mice immunisedwith the pCI::mCTLA4-hIg-HA construct had comparable levels of antiviralantibody to mice given the split virus vaccine (p=0.97). The antiviralantibody titres of sera collected on day 54 post-priming were alsodetermined (FIG. 15). Overall, the day 54 titres were similar to thosemeasured in sera collected on day 14, and the level of antibody in theday 54 sera of mice given the pCI::mCTLA4-hIg-HA construct remainedsignificantly higher than that of mice given pCI::CD5L-hIg-HA (p=0.009)or pCI::CD5L-hIg-SIINFEKL (p=0.0028), and comparable to split virus(p=0.85).

[0106] Ability of PR8 HA Constructs to Elicit Protective Immunity

[0107] Protection of vaccinated mice from influenza infection wasassessed by examining the ability of mice to clear a challenge dose ofvirus from their lungs by five days post-infection. It should be notedthat both antibody and cytotoxic T cell-mediated responses can lead toviral titre reduction within this 5 day interval. Mice were challengedon day 65 post-priming and the titre of virus in their lung wasdetermined by a plaque assay. FIG. 16 shows that all mice immunised withinfectious virus were able to clear the challenge dose of virus. Of themice given DNA constructs, the lung virus titres of mice immunised withthe pCI::mCTLA4-hIg-HA construct were significantly lower than those ofmice immunised with either pCI::CD5L-hIg-HA (p=0.0004) orpCI::CD5L-hIg-SIINFEKL (p =0.0002). Also the level of clearance observedin the pCI::mCTLA4-hIg-HA construct-immunised mice was almost as good asthat seen in mice given the split virus vaccine.

[0108] Conclusions pCI::CTLA4-hIg-HA conferred higher antibody levelsand better protection against challenge compared with the control vectorpCI::CD5L-hIg-HA demonstrating the immune enhancing effect of theincorporation of the targeting molecule.

EXAMPLE 5

[0109] Use of CTLA4 in Corynebacterium pseudotuberculosis in Sheep.

[0110] Introduction

[0111]Corynebacterium pseudotuberculosis is the causative agent ofcaseous lymphadenitis (CLA) in sheep. Established infection by thesebacteria leads to the formation of abscesses in the lymph nodes,especially the draining lymph node of the site of infection.Phospholipase D (PLD) has been characterised as a virulence factor and aprotective antigen for CLA. Indeed formalin-treated PLD²⁰ or geneticallytoxoided PLD (ΔPLD²²) has been shown to protect sheep from CLA.

[0112] We have used the genetically toxoided PLD as the basis for ourDNA vaccination approach and investigated whether the addition of bovine(b)CTLA4-hIg or hIg alone to the APLD construct enhanced the immuneresponse to PLD and to hIg.

[0113] Materials and Methods.

[0114] DNA Constructs

[0115] Using the known sequence of bCTLA4 (GenBank accession numberX15070), the bCTLA4 gene was isolated from bovine peripheral bloodmononuclear cells. A PCR product of bCTLA-4 (729 bp) was cloned into theZeroblunt TM cloning vector according to the manufacturer's instructions(Invitrogen) and sequenced using the Applied Biosystems automatedsequencer. The sequence of bCTLA4 was found to be identical to thepublished sequence.

[0116] The following constructs were generated in the pCI vector for DNAinmunisation:

[0117] pCI::bCTLA4-hIg-ΔPLD

[0118] pCI::ΔPLD

[0119] pCI::bCTLA4-hIg

[0120] pCI::CD5L-hIg-ΔPLD*

[0121] *CD5L refers to the leader sequence of the CD5 molecule allowingthe hulg-ΔPLD protein to be secreted.

[0122] Experimental Animals and Immunisation Regimen

[0123] Cross-bred ewes aged 12 weeks were used in the challenge trial.10 animals were allocated randomly to each group. Animals werepre-screened for the presence of antibodies to PLD and toCorynebacterium pseudotuberculosis lysate. Positive animals wereexcluded from the trial. Shearing, vaccination and tail docking ofanimals was avoided to minimise risk of infection with Corynebacteriumpseudotuberculosis.

[0124] Animals were injected intra-muscularly with 500 μg LPS free pCIplasmid DNA (coding for either pCI::bCTLA4-hIg-ΔPLD, pCI::ΔPLD,pCI::bCTLA4-huIg or pCI::CD5L-huIg-ΔPLD) in 5 ml of PBS. Control animalsreceived either Glanvac or were left un-immunised. All animals receivedthe same vaccine, at the same dose, 4 weeks later.

[0125] Challenge

[0126] Bacterial cultures of wild type Corynebacteriumpseudotuberculosis were grown at 37 ° C. in Brain heart infusion broth(Difco Laboratories) containing 0. 1% Tween 80 (BHI).

[0127] All sheep were challenged 6 weeks after primary immunisationusing a 1 ml dose of 10⁶ CFU of Corynebactefium pseudotuberculosisinjected just above the coronet of the right hind lateral claw.

[0128] Immunological Assays

[0129] Sera were collected from the sheep at weekly intervals andassayed for the presence of antibodies to genetically detoxified PLD(ΔPLD) and hIg using an ELISA. Plates were coated with {fraction (1/50)}of culture supernatant from APLD expressing Corynebacteriumpseudotuberculosis or 5 g/ml hMg protein. The sera were diluted in twofold steps starting at {fraction (1/100)} and {fraction (1/10)} for thedetection of anti-ΔPLD and anti-hIg antibodies respectively. Titres werecalculated by linear regression on a double logarithmic scale in thelinear part of the graph. The titre was defined as the dilution, whichresulted in an O.D. 0.3 in the ELISA.

[0130] T cell proliferation assays were performed in triplicates using 2concentrations of ΔPLD ({fraction (1/50)} and {fraction (1/250)}) or hIg(5 g/ml and 25 g/ml) as an antigen. PBMC were purified by ficollgradient and cultured in vitro for 3 days. The cultures were pulsed with3H-methyl-thymidine for 18 hours before being harvested on glass fibrefilters and radioactive incorporation assessed. The results arepresented as stimulation indices (i.e. ratio between counts obtainedwith antigen over counts obtained without antigen).

[0131] Statistical analysis was performed using the Systat program. Thenon-parametric Mann-Whitney U test was used to calculate significance. pvalues below 0.05 were considered significant.

[0132] Results

[0133] Anti-hIg Antibody Levels

[0134] The antibody titres to human immunoglobulin (hIg) reflect theimmune response to the DNA vaccination against the hIg part of thefusion protein in the case of bCTLA4-hIg-ΔPLD, CD5L-hIg-ΔPLD andbCTLA4-hIg. By comparing the response to hIg from the animals injectedwith pCI::bCTLA4-hIg-ΔPLD to these injected with pCl::CD5L-hIg-ΔPLD itis possible to specifically evaluate the effect of bCTLA4 targeting onthe immunogenicity of hIg. Results shown in FIG. 17 indicate that theantibody response to hIg in animals injected with pCI::bCTLA4-hIg-ΔPLD(filled squares) is both earlier and stronger than the anti-hIg responseinduced in animals immunised with pCI::CD5L-hIg-ΔPLD (closed circles).The Mann-Whitney U test indicates a statistically significant differencefor weeks 3 and 4. Corroborating these results the anti-hIg response inthe group injected with pCI::bCTLA4-hIg (closed triangles) is alsoearlier and stronger than the response in animals injected withpCI::CD5L-hIg-ΔPLD.

[0135] Anti-ΔPLD Antibody Levels

[0136] Immunisation with the detoxified-PLD protein antigen (Glan-Vac)resulted in little or no detectable antibodies during the first 7 weeksafter immunisation. Two weeks after challenge antibody levels increaseddramatically. This is consistent with previously reported results²¹

[0137] All groups imnmunised with pCI encoding the ΔPLD antigen eitheralone or as a fusion protein with bCTLA4-hIg or with CD5L-hIg, resultedin similar kinetics of antibody production. Indeed, no significantanti-ΔPLD antibody levels were detected until 2 weeks post-challenge(i.e. week 8). At each time point there was no significant differencebetween the level of antibodies induced by the different pCI constructs,indicating that all constructs have a similar ability to induce immunememory to ΔPLD. However, this result is not surprising in light of thefact that detoxified-PLD is not a classical antigen in that two doses ofthe protein antigen (Glan-Vac group) failed to induce substantialantibody levels. This result has also been reported by others²². Thusdetoxified-PLD seems to result in the induction of immune memory withoutinduction of high antibody titres. This immune memory is then activatedduring the early stage of the challenge by PLD expressed byCorynebacterium pseudotuberculosis. It is interesting to note that ΔPLDexpressed in eukaryotic cells (COSm6 cells) has a slightly larger sizecompared to ΔPLD expressed in Corynebacterium pseudotuberculosis (FIG.18). This difference can be accounted for by the possible glycosylationof ΔPLD in eukaryotic cells. As in the case with anti-hIg antibodylevels it is noted that sheep, in contrast to mice, do not produceantibodies over prolonged periods of time following DNA vaccination.

[0138] T Cell Responses to APLD and hIg.

[0139] T cell responses to ΔPLD and hIg were analysed at 3 weeks, 6weeks and 9 weeks. No significant antigen specific proliferation, tothese two antigens could be demonstrated in peripheral blood mononuclearcells (PBMC) at any time point. This is most likely due to technicaldifficulties since the kinetics of the anti ΔPLD response indicate thelikely presence of memory T cells in the vaccinated animals.

[0140] Anti-ΔPLD Response Subsequent to Challenge with Virulent C.pseudotuberculosis.

[0141] Challenge was performed at 6 weeks post-immunisation (i.e. twoweeks post-booster immunisation). Observation of the site of inoculationat week 10 did not reveal any correlation with immunisation regime.Except in the case of the unvaccinated control and pCI::bCTLA4-hIgimmunised animals, the antibody response to ΔPLD increased two weekspost-challenge (week 8). This indicates that the immune memory inducedby the immunisation with DNA can be boosted with ΔPLD produced byCorynebacterium pseudotuberculosis. This indicates that although ΔPLDproduced by DNA vaccination is most likely glycosylated there is stillcross-reaction with bacterial wild-type PLD.

[0142] In the un-immunised control animals the level of anti-ΔPLDantibodies remains low until 3 to 4 weeks post-challenge (week 9). Thiskinetics of antibody appearance has been described previously²².

[0143] Interestingly the antibody levels in the animals primed with ΔPLDby DNA vaccination or detoxified-PLD by Glan-Vac injection, diminishedafter week 8 suggesting that the PLD antigen is no longer boosting theimmune response. This may be due to a diminished level of PLD secretiondue to the animals clearing Corynebacterium pseudotuberculosis.

[0144] Protection from Challenge with Virulent Corynebacteriumpseudotuberculosis.

[0145] At week 12 post vaccination (6 weeks post challenge) necropsy ofall animals was performed to evaluate the protective efficacy of theimmunisation protocol. Both left and right popliteal, inguinal andprefemoral lymph nodes were dissected and visually assessed for thecharacteristic abscesses induced by Corynebacterium pseudotuberculosis.The lungs were palpated to detect abscesses and no lung abscesses werefound in any of the animals. Protection was defined as the absence ofthe characteristic Corynebacterium pseudotuberculosis abscesses in anyof the lymph nodes. In two sheep small dry lesions were observed in thedraining popliteal lymph node, clearly distinct from the abscesses inthe other animals. These lesions were most likely foci ofCorynebacterium pseudotuberculosis which were regressing in the face ofan effective immune response and these animals were also scored as“protected”. From FIG. 19 it can be seen that while about 10% of theunvaccinated animals did not develop lesions, 90% were protected byvaccination with Glan-Vac. Only I animal out of 10 injected withpCI::CTLA4-hIg did not have abscesses in the lymph nodes. All animalsvaccinated with DNA encoding for ΔPLD (pCI::bCTLA4-hIg-ΔPLD, pCI::ΔPLDand pCI::CD5L-hIg-ΔPLD) were afforded some protection. The level ofprotection ranged from 40 to 70% with the highest degree of protectionobserved in the group injected with pCI::CTLA4-hIg-ΔPLD.

[0146] Conclusion

[0147] This study has examined the ability of bCTLA4 to increase theimmune response to antigens during DNA immunisation in sheep. bCTLA4 hasthe ability to accelerate and increase the immune response to hIg.Challenge with Corynebacterium pseudotuberculosis indicated that DNAvaccination could induce a protective immune response. When comparingthe protection obtained with pCI::bCTLA4-hIg-ΔPLD and pCI::CD5L-hIg-APLD a substantial difference was observed.

[0148] The protection induced by CD5L-hIg-ΔPLD is lower than theprotection induced by ΔPLD on its own. This difference may be due to thefact that the ΔPLD molecule is much smaller than the bCD5L-hIg-ΔPLD andhence could be expressed in an acceptable conformation more easily. Onecould therefore expect that the bCTLA4-hIg-ΔPLD molecule which is evenlarger would be even more difficult to express. Hence the fact that thebCTLA4-hIg-ΔPLD molecule induced higher levels of protection indicatesthat the CTLA4 molecule effectively increases the immunogenicity of thefusion protein. It can be reasonably expected that by improving thelevel of expression and folding of the molecule even better protectioncould be obtained. One way of achieving this would be to reduce the sizeof the hIg portion of the fusion protein.

EXAMPLE 7

[0149] Vaccination of C3H/He Mice with DNA Encoding the Parasite SurfaceAntigen 2 (PSA2) of Leishmania major.

[0150] Introduction

[0151] Protection against infection with this obligatory intracellularparasite is provided by CD4+ T cells of the macrophage activating, Th1type. We have shown that injection of plasmid DNA encoding full lengthPSA2 induced significant protection against a challenge infection inC3H/He mice. This protection correlated with the induction of a verylow, but consistent Th1 type of immune response, ie induction of T cellssecreting interferon gamma, but no IL-4. Here we aim to examine theability of CTLA4-Ig to improve the level of protection.

[0152] Materials and methods

[0153] PSA2 encoding residues 33-357 (missing the leader and gpi signal)was fused C-terminal to either pCI::CD5L-hIg or pCI::mCTLA4-hIg. DNAencoding a secreted form of PSA2 (residues 1-357) was also made(pCI::PSA2)

[0154] Groups of 8 mice were injected with 100 μg DNA in 100 μlphosphate buffered saline (PBS) intramuscularly twice at two weekintervals. Two weeks after each injection the mice were bled and theserum tested for antibodies to PSA-2. Two weeks after the secondinjection the mice were infected intradermally with 100,000promastigotes. The development of lesions at the site of infection wasmonitored weekly and scored according to size. Parasite burdens in thelymph nodes draining the lesion were determined by limiting dilutionanalysis at 7 weeks after challenge infection.

[0155] Results

[0156] Antibody Production

[0157] Antibodies were measured only by ELISA OD at a single point. Twoweeks after the first immunisation, 4 of 8 mice immunised withpCI::mCTLA4-hIg-PSA2 and 3 of 8 mice given pCI::CD5L-hIg-PSA2 producedsignificant antibody at a dilution of 1:500. However, after the secondinjection of DNA mice immunised with pCI::mCTLA4-hIg-PSA2,pCI::CD5L-hIg-PSA2 and our own secreted form of PSA-2 showed significantlevels of antibody at this dilution. The PBS control had backgroundantibody.

[0158] Protection from Infection

[0159] Mice immunised with DNA encoding pCI::CD5L-hIg-PSA2 and thecontrols PBS and vector DNA developed lesions at the site of infection 1week after challenge. Mice immunised with pCI::mCTLA4-hIg-PSA2 orpCI::PSA2 developed lesions only 3 weeks after infection and the size ofthe lesions was smaller compared to the rest. Mice immunised withpCI::mCTLA4-hIg-PSA2 or pCI::PSA2 also had the smallest number of micewhich developed lesions with only 5 of 8 mice showing any lesions at thepeak of the disease curve (FIG. 20). Notably, pCI::mCTLA4-hIg-PSA2conferred better protection than pCI::CD5L-hIg-PSA2 (p=0.0001; log ranktest).

Summary

[0160] The ability to overcome the problem of low or absentresponsiveness in DNA immunisation by antigen targeting enhances thepotential of genetic vaccines. The present inventors also show thatintramuscular injection of DNA can also be employed to deviate immuneresponses to the same antigen allowing for the development of vaccinesin which the response most likely to confer protection can be generated.

[0161] Intramuscular injection of expression plasmids shows greatpotential for genetic vaccination. The present inventors have shown thatfusion proteins consisting of antigen and cell surface receptor ligandscould deliver antigen to sites of immune induction which enhance theimmune response and possibly the efficacy of genetic vaccines. As setout above mice were immunized with plasmids encoding Fc fragment ofhuman IgG1 as antigen. This Ig fragment was fused with CTLA4 (CTLA4Ig)for delivery to antigen presenting cells (APC) expressing B-7, or withL-selectin (L-SELIg) for delivery to high endothelial venule cells oflymph nodes. L-selectin binds CD34 and MadCAM-1 and so could target anylymphoid organ with these receptors (12). Enhanced antibody responseswere shown in both the CTLA4Ig and L-SELIg immunized mice, 1000 and 100fold respectively at 4 weeks. Moreover the response after CTLA4Igimmunisation was the most rapid. Immune deviation from an IgG2a to anIgG1 dominated response occurred in CTLA4Ig immunized mice and allowsfor the development of genetic vaccines in which the response mostlikely to confer protection can be generated.

[0162] It will be appreciated by persons skilled in the art thatnumerous variations and/or modifications may be made to the invention asshown in the specific embodiments without departing from the spirit orscope of the invention as broadly described. The present embodimentsare, therefore, to be considered in all respects as illustrative and notrestrictive.

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1 1 1 11265 DNA Murine 1 tctagagtcg accaattctc atgtttgaca gcttatcatcgcagatcctg agcttgtatg 60 gtgcactctc agtacaatct gctctgctgc cgcatagttaagccagtatc tgctccctgc 120 ttgtgtgttg gaggtcgctg agtagtgcgc gagcaaaatttaagctacaa caaggcaagg 180 cttgaccgac aattgcatga agaatctgct tagggttaggcgttttgcgc tgcttcgcga 240 tgtacgggcc agatatacgc gtatctgagg ggactagggtgtgtttaggc gcccagcggg 300 gcttcggttg tacgcggtta ggagtcccct caggatatagtagtttcgct tttgcatagg 360 gagggggaaa tgtagtctta tgcaatacac ttgtagtcttgcaacatggt aacgatgagt 420 tagcaacatg ccttacaagg agagaaaaag caccgtgcatgccgattggt ggaagtaagg 480 tggtacgatc gtgccttatt aggaaggcaa cagacaggtctgacatggat tggacgaacc 540 actgaattcc gcattgcaga gataattgta tttaagtgcctagctcgata caataaacgc 600 catttgacca ttcaccacat tggtgtgcac ctccaagctgggtaccagct gctagcaagc 660 ttgctagcgg ccgctcgagc tcagcacatt tgccccccagccatggcttg tcttggactc 720 cggaggtaca aagctcaact gcagctgcct tctaggacttggccttttgt agccctgctc 780 actcttcttt tcatcccagt cttctctgaa gccatacaggtgacccaacc ttcagtggtg 840 ttggctagca gccatggtgt cgccagcttt ccatgtgaatattcaccatc acacaacact 900 gatgaggtcc gggtgactgt gctgcggcag acaaatgaccaaatgactga ggtctgtgcc 960 acgacattca cagagaagaa tacagtgggc ttcctagattaccccttctg cagtggtacc 1020 tttaatgaaa gcagagtgaa cctcaccatc caaggactgagagctgttga cacgggactg 1080 tacctctgca aggtggaact catgtaccca ccgccatactttgtgggcat gggcaacggg 1140 acgcagattt atgtcattga tccagaacca tgcccggattctggtaagta taagcttcag 1200 cgctcctgcc tggacgcatc ccggctatgc agccccagtccagggcagca aggcaggccc 1260 cgtctgcctc ttcacccgga gcctctgccc gccccactcatgctcaggga gagggtcttc 1320 tggctttttc ccaggctctg ggcaggcaca ggctaggtgcccctaaccca ggccctgcac 1380 acaaaggggc aggtgctggg ctcagacctg ccaagagccatatccgggag gaccctgccc 1440 ctgacctaag cccaccccaa aggccaaact ctccactccctcagctcgga caccttctct 1500 cctcccagat tccagtaact cccaatcttc tctctgcagagcccaaatct tgtgacaaaa 1560 ctcacacatg cccaccgtgc ccaggtaagc cagcccaggcctcgccctcc agctcaaggc 1620 gggacaggtg ccctagagta gcctgcatcc agggacaggccccagccggg tgctgacacg 1680 tccacctcca tctcttcctc agcacctgaa ctcctggggggaccgtcagt cttcctcttc 1740 cccccaaaac ccaaggacac cctcatgatc tcccggacccctgaggtcac atgcgtggtg 1800 gtggacgtga gccacgaaga ccctgaggtc aagttcaactggtacgtgga cggcgtggag 1860 gtgcataatg ccaagacaaa gccgcgggag gagcagtacaacagcacgta ccgggtggtc 1920 agcgtcctca ccgtcctgca ccaggactgg ctgaatggcaaggagtacaa gtgcaaggtc 1980 tccaacaaag ccctcccagc ccccatcgag aaaaccatctccaaagccaa aggtgggacc 2040 cgtggggtgc gagggccaca tggacagagg ccggctcggcccaccctctg ccctgagagt 2100 gaccgctgta ccaacctctg tcctacaggg cagccccgagaaccacaggt gtacaccctg 2160 cccccatccc gggatgagct gaccaagaac caggtcagcctgacctgcct ggtcaaaggc 2220 ttctatccca gcgacatcgc cgtggagtgg gagagcaatgggcagccgga gaacaactac 2280 aagaccacgc ctcccgtgct ggactccgac ggctccttcttcctctacag caagctcacc 2340 gtggacaaga gcaggtggca gcaggggaac gtcttctcatgctccgtgat gcatgaggct 2400 ctgcacaacc actacacgca gaagagcctc tccctgtctccgggtaaatg agtgcgacgg 2460 ccggatccag acatgataag atacattgat gagtttggacaaaccacaac tagaatgcag 2520 tgaaaaaaat gctttatttg tgaaatttgt gatgctattgctttatttgt aaccattata 2580 agctgcaata aacaagttaa caacaacaat tgcattcattttatgtttca ggttcagggg 2640 gaggtgggga ggttttttaa agcaagtaaa acctctacaaatgtggtatg gctgattatg 2700 atccggctgc ctcgcgcgtt tcggtgatga cggtgaaaacctctgacaca tgcagctccc 2760 ggagacggtc acagcttgtc tgtaagcgga tgccgggagcagacaagccc gtcagggcgc 2820 gtcagcgggt gttggcgggt gtcggggcgc agccatgaccggtcgaccac tgggcgccag 2880 aaatccgcgc ggtggttttt gggggtcggg ggtgtttggcagccacagac gcccggtgtt 2940 cgtgtcgcgc cagtacatgc ggtccatgcc caggccatccaaaaaccatg ggtctgtctg 3000 ctcagtccag tcgtggacca gaccccacgc aacgcccaaaataataaccc ccacgaacca 3060 taaaccattc cccatggggg accccgtccc taacccacggggccagtggc tatggcaggg 3120 cctgccgccc cgacgttggc tgcgagccct gggccttcacccgaacttgg ggggtggggt 3180 ggggaaaagg aagaaacgcg ggcgtattgg ccccaatggggtctcggtgg ggtatcgaca 3240 gagtgccagc cctgggaccg aaccccgcgt ttatgaacaaacgacccaac acccgtgcgt 3300 tttattctgt ctttttattg ccgtcatagc gcgggttccttccggtattg tctccttccg 3360 tgtttcagtt agcctccccc atctccccta ttcctttgccctcggacgag tgctggggcg 3420 tcggtttcca ctatcggcga gtacttctac acagccatcggtccagacgg ccgcgcttct 3480 gcgggcgatt tgtgtacgcc cgacagtccc ggctccggatcggacgattg cgtcgcatcg 3540 accctgcgcc caagctgcat catcgaaatt gccgtcaaccaagctctgat agagttggtc 3600 aagaccaatg cggagcatat acgcccggag ccgcggcgatcctgcaagct ccggatgcct 3660 ccgctcgaag tagcgcgtct gctgctccat acaagccaaccacggcctcc agaagaagat 3720 gttggcgacc tcgtattggg aatccccgaa catcgcctcgctccagtcaa tgaccgctgt 3780 tatgcggcca ttgtccgtca ggacattgtt ggagccgaaatccgcgtgca cgaggtgccg 3840 gacttcgggg cagtcctcgg cccaaagcat cagctcatcgagagcctgcg cgacggacgc 3900 actgacggtg tcgtccatca cagtttgcca gtgatacacatggggatcag caatcgcgca 3960 tatgaaatca cgccatgtag tgtattgacc gattccttgcggtccgaatg ggccgaaccc 4020 gctcgtctgg ctaagatcgg ccgcagcgat cgcatccatggcctccgcga ccggctgcag 4080 aacagcgggc agttcggttt caggcaggtc ttgcaacgtgacaccctgtg cacggcggga 4140 gatgcaatag gtcaggctct cgctgaattc cccaatgtcaagcacttccg gaatcgggag 4200 cgcggccgat gcaaagtgcc gataaacata acgatctttgtagaaaccat cggcgcagct 4260 atttacccgc aggacatatc cacgccctcc tacatcgaagctgaaagcac gagattcttc 4320 gccctccgag agctgcatca ggtcggagac gctgtcgaacttttcgatca gaaacttctc 4380 gacagacgtc gcggtgagtt caggcttttt catatctcattgcccgggat ctgcggcacg 4440 ctgttgacgc tgttaagcgg gtcgctgcag ggtcgctcggtgttcgaggc cacacgcgtc 4500 accttaatat gcgaagtgga cctgggaccg cgccgccccgactgcatctg cgtgttcgaa 4560 ttcgccaatg acaagacgct gggcggggtt tgtgtcatcatagaactaaa gacatgcaaa 4620 tatatttctt ccggggacac cgccagcaaa cgcgagcaacgggccacggg gatgaagcag 4680 ggcatggcgg ccgacgcgct gggctacgtc ttgctggcgttcgcgacgcg aggctggatg 4740 gccttcccca ttatgattct tctcgcttcc ggcggcatcgggatgcccgc gttgcaggcc 4800 atgctgtcca ggcaggtaga tgacgaccat cagggacagcttcaaggatc gctcgcggct 4860 cttaccagcc taacttcgat cactggaccg ctgatcgtcacggcgattta tgccgcctcg 4920 gcgagcacat ggaacgggtt ggcatggatt gtaggcgccgccctatacct tgtctgcctc 4980 cccgcgttgc gtcgcggtgc atggagccgg gccacctcgacctgaatgga agccggcggc 5040 acctcgctaa cggattcacc actccaagaa ttggagccaatcaattcttg cggagaactg 5100 tgaatgcgca aaccaaccct tggcagaaca tatccatcgcgtccgccatc tccagcagcc 5160 gcacgcggcg cagcaaaagg ccaggaaccg taaaaaggccgcgttgctgg cgtttttcca 5220 taggctccgc ccccctgacg agcatcacaa aaatcgacgctcaagtcaga ggtggcgaaa 5280 cccgacagga ctataaagat accaggcgtt tccccctggaagctccctcg tgcgctctcc 5340 tgttccgacc ctgccgctta ccggatacct gtccgcctttctcccttcgg gaagcgtggc 5400 gctttctcat agctcacgct gtaggtatct cagttcggtgtaggtcgttc gctccaagct 5460 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgcgccttatccg gtaactatcg 5520 tcttgagtcc aacccggtaa gacacgactt atcgccactggcagcagcca ctggtaacag 5580 gattagcaga gcgaggtatg taggcggtgc tacagagttcttgaagtggt ggcctaacta 5640 cggctacact agaaggacag tatttggtat ctgcgctctgctgaagccag ttaccttcgg 5700 aaaaagagtt ggtagctctt gatccggcaa acaaaccaccgctggtagcg gtggtttttt 5760 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatctcaagaagatc ctttgatctt 5820 ttctacgggg tctgacgctc agtggaacga aaactcacgttaagggattt tggtcatgag 5880 attatcaaaa aggatcttca cctagatcct tttaaattaaaaatgaagtt ttaaatcaat 5940 ctaaagtata tatgagtaaa cttggtctga cagttaccaatgcttaatca gtgaggcacc 6000 tatctcagcg atctgtctat ttcgttcatc catagttgcctgactccccg tcgtgtagat 6060 aactacgata cgggagggct taccatctgg ccccagtgctgcaatgatac cgcgagaccc 6120 acgctcaccg gctccagatt tatcagcaat aaaccagccagccggaaggg ccgagcgcag 6180 aagtggtcct gcaactttat ccgcctccat ccagtctattaattgttgcc gggaagctag 6240 agtaagtagt tcgccagtta atagtttgcg caacgttgttgccattgctg caggcatcgt 6300 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctccggttcccaac gatcaaggcg 6360 agttacatga tcccccatgt tgtgcaaaaa agcggttagctccttcggtc ctccgatcgt 6420 tgtcagaagt aagttggccg cagtgttatc actcatggttatggcagcac tgcataattc 6480 tcttactgtc atgccatccg taagatgctt ttctgtgactggtgagtact caaccaagtc 6540 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgcccggcgtcaa cacgggataa 6600 taccgcgcca catagcagaa ctttaaaagt gctcatcattggaaaacgtt cttcggggcg 6660 aaaactctca aggatcttac cgctgttgag atccagttcgatgtaaccca ctcgtgcacc 6720 caactgatct tcagcatctt ttactttcac cagcgtttctgggtgagcaa aaacaggaag 6780 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaatgttgaatac tcatactctt 6840 cctttttcaa tattattgaa gcatttatca gggttattgtctcatgagcg gatacatatt 6900 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgcacatttcccc gaaaagtgcc 6960 acctgacgtc taagaaacca ttattatcat gacattaacctataaaaata ggcgtatcac 7020 gaggcccttt cgtcttcaag aattctcatg tttgacagcttatcatcgat aagctgatcc 7080 tcacaggccg cacccagctt ttcttccgtt gccccagtagcatctctgtc tggtgacctt 7140 gaagaggaag aggaggggtc ccgagaatcc ccatccctaccgtccagcaa aaagggggac 7200 gaggaatttg aggcctggct tgaggctcag gacgcaaatcttgaggatgt tcagcgggag 7260 ttttccgggc tgcgagtaat tggtgatgag gacgaggatggttcggagga tggggaattt 7320 tcagacctgg atctgtctga cagcgaccat gaaggggatgagggtggggg ggctgttgga 7380 gggggcagga gtctgcactc cctgtattca ctgagcgtcgtctaataaag atgtctattg 7440 atctctttta gtgtgaatca tgtctgacga ggggccaggtacaggacctg gaaatggcct 7500 aggagagaag ggagacacat ctggaccaga aggctccggcggcagtggac ctcaaagaag 7560 agggggtgat aaccatggac gaggacgggg aagaggacgaggacgaggag gcggaagacc 7620 aggagccccg ggcggctcag gatcagggcc aagacatagagatggtgtcc ggagacccca 7680 aaaacgtcca agttgcattg gctgcaaagg gacccacggtggaacaggag caggagcagg 7740 agcgggaggg gcaggagcag gaggggcagg agggaggccggggtcgagga ggtagtggag 7800 gccggggtcg aggaggtagt ggaggccgcc ggggtagaggacgtgaaaga gccagggggg 7860 gaagtcgtga aagagccagg gggagaggtc gtggacgtggagaaaagagg cccaggagtc 7920 ccagtagtca gtcatcatca tccgggtctc caccgcgcaggccccctcca ggtagaaggc 7980 catttttcca ccctgtaggg gaagccgatt attttgaataccaccaagaa ggtggcccag 8040 atggtgagcc tgacgtgccc ccgggagcga tagagcagggccccgcagat gacccaggag 8100 aaggcccaag cactggaccc cggggtcagg gtgatggaggcaggcgcaaa aaaggagggt 8160 ggtttggaaa gcatcgtggt caaggaggtt ccaacccgaaatttgagaac attgcagaag 8220 gtttaagagc tctcctggct aggagtcacg tagaaaggactaccgacgaa ggaacttggg 8280 tcgccggtgt gttcgtatat ggaggtagta agacctccctttacaaccta aggcgaggaa 8340 ctgcccttgc tattccacaa tgtcgtctta caccattgagtcgtctcccc tttggaatgg 8400 cccctggacc cggcccacaa cctggcccgc taagggagtccattgtctgt tatttcatgg 8460 tctttttaca aactcatata tttgctgagg ttttgaaggatgcgattaag gaccttgtta 8520 tgacaaagcc cgctcctacc tgcaatatca gggtgactgtgtgcagcttt gacgatggag 8580 tagatttgcc tccctggttt ccacctatgg tggaaggggctgccgcggag ggtgatgacg 8640 gagatgacgg agatgaagga ggtgatggag atgagggtgaggaagggcag gagtgatgta 8700 acttgttagg agacgccctc aatcgtatta aaagccgtgtattcccccgc actaaagaat 8760 aaatccccag tagacatcat gcgtgctgtt ggtgtatttctggccatctg tcttgtcacc 8820 attttcgtcc tcccaacatg gggcaattgg gcatacccatgttgtcacgt cactcagctc 8880 cgcgctcaac accttctcgc gttggaaaac attagcgacatttacctggt gagcaatcag 8940 acatgcgacg gctttagcct ggcctcctta aattcacctaagaatgggag caaccagcat 9000 gcaggaaaag gacaagcagc gaaaattcac gcccccttgggaggtggcgg catatgcaaa 9060 ggatagcact cccactctac tactgggtat catatgctgactgtatatgc atgaggatag 9120 catatgctac ccggatacag attaggatag catatactacccagatatag attaggatag 9180 catatgctac ccagatatag attaggatag cctatgctacccagatataa attaggatag 9240 catatactac ccagatatag attaggatag catatgctacccagatatag attaggatag 9300 cctatgctac ccagatatag attaggatag catatgctacccagatatag attaggatag 9360 catatgctat ccagatattt gggtagtata tgctacccagatataaatta ggatagcata 9420 tactacccta atctctatta ggatagcata tgctacccggatacagatta ggatagcata 9480 tactacccag atatagatta ggatagcata tgctacccagatatagatta ggatagccta 9540 tgctacccag atataaatta ggatagcata tactacccagatatagatta ggatagcata 9600 tgctacccag atatagatta ggatagccta tgctacccagatatagatta ggatagcata 9660 tgctatccag atatttgggt agtatatgct acccatggcaacattagccc accgtgctct 9720 cagcgacctc gtgaatatga ggaccaacaa ccctgtgcttggcgctcagg cgcaagtgtg 9780 tgtaatttgt cctccagatc gcagcaatcg cgcccctatcttggcccgcc cacctactta 9840 tgcaggtatt ccccggggtg ccattagtgg ttttgtgggcaagtggtttg accgcagtgg 9900 ttagcggggt tacaatcagc caagttatta cacccttattttacagtcca aaaccgcagg 9960 gcggcgtgtg ggggctgacg cgtgccccca ctccacaatttcaaaaaaaa gagtggccac 10020 ttgtctttgt ttatgggccc cattggcgtg gagccccgtttaattttcgg gggtgttaga 10080 gacaaccagt ggagtccgct gctgtcggcg tccactctctttccccttgt tacaaataga 10140 gtgtaacaac atggttcacc tgtcttggtc cctgcctgggacacatctta ataaccccag 10200 tatcatattg cactaggatt atgtgttgcc catagccataaattcgtgtg agatggacat 10260 ccagtcttta cggcttgtcc ccaccccatg gatttctattgttaaagata ttcagaatgt 10320 ttcattccta cactagtatt tattgcccaa ggggtttgtgagggttatat tggtgtcata 10380 gcacaatgcc accactgaac cccccgtcca aattttattctgggggcgtc acctgaaacc 10440 ttgttttcga gcacctcaca tacaccttac tgttcacaactcagcagtta ttctattagc 10500 taaacgaagg agaatgaaga agcaggcgaa gattcaggagagttcactgc ccgctccttg 10560 atcttcagcc actgcccttg tgactaaaat ggttcactaccctcgtggaa tcctgacccc 10620 atgtaaataa aaccgtgaca gctcatgggg tgggagatatcgctgttcct taggaccctt 10680 ttactaaccc taattcgata gcatatgctt cccgttgggtaacatatgct attgaattag 10740 ggttagtctg gatagtatat actactaccc gggaagcatatgctacccgt ttagggttaa 10800 caagggggcc ttataaacac tattgctaat gccctcttgagggtccgctt atcggtagct 10860 acacaggccc ctctgattga cgttggtgta gcctcccgtagtcttcctgg gcccctggga 10920 ggtacatgtc ccccagcatt ggtgtaagag cttcagccaagagttacaca taaaggcaat 10980 gttgtgttgc agtccacaga ctgcaaagtc tgctccaggatgaaagccac tcagtgttgg 11040 caaatgtgca catccattta taaggatgtc aactacagtcagagaacccc tttgtgtttg 11100 gtcccccccc gtgtcacatg tggaacaggg cccagttggcaagttgtacc aaccaactga 11160 agggattaca tgcactgccc cgaatacaaa acaaaagcgctcctcgtacc agcgaagaag 11220 gggcagagat gccgtagtca ggtttagttc gtccggcggcggggc 11265

1. A DNA molecule for use in raising an immune response to an antigen,the DNA molecule including a first sequence encoding a targetingmolecule, a second sequence encoding the antigen or an epitope thereof,and optionally a third sequence encoding a polypeptide which promotesdimerisation or multimerisation of the product encoded by the DNAmolecule.
 2. A DNA molecule as claimed in claim 1 in which the antigenor epitope thereof encoded by the second sequence is a polypeptide whichpromotes dimerisation or multimerisation of the product encoded by theDNA molecule and the third sequence is absent.
 3. A DNA molecule asclaimed in claim 1 in which the third sequence is present.
 4. A DNAmolecule as claimed in any one of claims 1 to 3 in which the targetingmolecule encoded by the first sequence is a ligand which targetslymphoid cells, lymphoid sites or antigen presenting cells.
 5. A DNAmolecule as claimed in any one of claims 1 to 4 in which the targetingmolecule encoded by the first sequence is selected from the groupconsisting of CTLA4, L-selectin, CD40L, OX40 and CD28, and antibodies toreceptors on antigen presenting cells.
 6. A DNA molecule as claimed inany one of claims 1 to 5 in which the targeting molecule encoded by thefirst sequence is CTLA4.
 7. A polypeptide, the polypeptide being encodedby the DNA molecule as claimed in any one of claims 1 to
 6. 8. A vectorincluding the DNA molecule as claimed in any one of claims 1 to
 6. 9. Acomposition for use in raising an immune response in an animal thecomposition including the DNA molecule as claimed in any one of claims 1to 6 or the vector as claimed in claim 8 and an acceptable diluent orexcipient.
 10. A composition for use in raising an immune response in ananimal the composition including the polypeptide as claimed in claim 7and an acceptable diluent or excipient.
 11. A method of raising animmune response in an animal, the method comprising administering to theanimal the composition as claimed in claim 9 or claim
 10. 12. A methodof deviating the immune response to an antigen in an individual, themethod comprising administering to the individual a DNA moleculeincluding a first sequence encoding CTLA4, a second sequence encodingthe antigen or an epitope thereof, and optionally a third sequenceencoding a polypeptide which promotes dimerisation or multimerisation ofthe product encoded by the DNA molecule.
 13. A method as claimed inclaim 12 in which the antigen or epitope thereof encoded by the secondsequence is a polypeptide which promotes dimerisation or multimerisationof the product encoded by the DNA molecule and the third sequence isabsent.
 14. A method as claimed in claim 12 in which the third sequenceis present.